This invention relates to tagged molecules (distinguishable from untagged, but otherwise identical, molecules), methods of preparing tagged molecules, nucleic acid sequences and constructs encoding tagged molcules, and a method of distinguishing between tagged and untagged (but otherwise identical) molecules.
In particular, the present invention relates to a method of tagging a protein with a therapeutically acceptable tag which enables detection of the tagged protein administered exogenously to humans, bovines or other animals where the same (but untagged) protein is produced endogenously, and a method of detecting and differentiating the tagged protein over the endogenous protein. In particular, the method is suitable for application to human growth hormone (hGH), to enable differential detection of exogenously administered hGH in humans, for example, to determine whether hGH is being administered unlawfully for its performance enhancing effects.
Previously, the usual method of differentiating exogenously administered protein from the endogenous one has been to tag the exogenous protein with radioactive labels. Because of the hazards of radioactivity, radioactively tagged proteins are administered to patients over short periods of time in controlled conditions and under medical supervision. Further, radioactive labels are not therapeutically acceptable since they are intrusive to the biological system in which such tagged proteins are administered. Other tagging methods tend to alter the biological function of the protein molecule and therefore are no longer suitable for therapeutic use. Such prior art tagging methods are therefore limited to controlled research uses and do not have widespread cost effective commercial applications.
Some amino acids, for example tryptophan (W) and tyrosine (Y) in particular, are natural fluorophores, which fluoresce when appropriately stimulated. This fluorescence can be detected and measured with standard prior art fluorescence detection techniques. Proteins which contain such fluorophores in their amino acid sequence may also fluoresce when appropriately stimulated. The level of fluorescence can be crudely related to the number of fluorophores in the protein. The fluorescent yield of any fluorophore is sensitive to its local environment such that, for example, there may be a difference between its fluorescence in an aqueous and a hydrophobic environment. Waldman et al (1987 Biochem. Biophys. Acta 931, 66-71; 1988 Biochem. Biophys Res. Comm. 150 (2), 752-759), Corinne (1991 Biochemistry 30, 1028-1036) and others have exploited this property to perform in vitro laboratory studies on conformational and structural changes of lactate dehydrogenase when, for example, substrate binding occurs. Waldman and Corrine have mutated lactate dehydrogenase to incorporate tryptophan residues at the substrate binding site. This technique is restricted to use as a research tool for conformational and structural studies of proteins in vitro, since often the full biological activity or structural conformation of the native protein is lost. Thus, such modified proteins are no longer suitable for therapeutic purposes and there is no disclosure or suggestion of pharmaceutical compositions comprising the mutated protein. Moreover, there is no disclosure or suggestion in the prior art that such mutations could form the basis for a method of distinguishing the altered compound from the naturally occurring compound.
WO 94/10200 discloses and is concerned with amino acid substitutions in somatotropin (i.e. Growth Hormone) which provide increased conformational and chemical stability.
There is no suggestion in WO 94/10200 that modifications can be made to Growth Hormone for the purpose of distinguishing between endogenous Growth Hormone present in a subject and exogenous Growth Hormone administered to the subject. A number of amino acid substitutions in somatotropin are disclosed or suggested in WO 94/10200 which, because of the natural fluorophore activity of the amino acid residues tryptophan and tyrosine (discussed above), result in a somatotropin molecule having an altered fluorescence activity relative to the wild type, unsubstituted molecule Such substitutions include the following:
G40xe2x86x92Y (i.e. glycine substituted by tyrosine at residue number 40); F52xe2x86x92Y: W86xe2x86x92F, Y, L, I or V; F103xe2x86x92Y; 1137xe2x86x92Y;
A reliable method for differentiating and detecting exogenously administered hGH is particularly desirable when attempting to monitor the pharmacokinetics and/or pharmacodynamics of hGH, or to detect its unlawful administration by athletes and others who illicitly use hGH for improving their performance. Presently, standard detection methods (e.g. HPLC, ELISA), are used for measuring the total amount of hGH in an athletes"" blood or urine samples, and by subtracting the expected levels referenced to the general population, estimations of elevated hGH levels can be made. However, as levels vary considerably between individuals, and exogenous levels fall rapidly with time, this is a very crude measurement. In addition, as the performance enhancing effects last much longer than the detectable transient elevated levels of hGH in these samples, unless samples are taken shortly after administration the technique does not give indisputable proof that exogenous hGH has or has not been used.
The present invention seeks to alleviate the above mentioned problems by tagging or modifying a protein (such as hGH) with a therapeutically acceptable tag which can be detected simply and can be differentiated from the endogenous protein present in a sample of cells, blood, urine or other body fluid. The invention has little or no effect on the biological activity of the protein, such that the modified protein can be administered therapeutically in the same manner as the unmodified protein. Thus, the modified or tagged protein can be safely prescribed by physicians for existing or new therapeutic purposes, and also economically manufactured commercially at substantially the same cost as the untagged protein.
A further advantage of the present invention is that although levels of the exogenous protein may drop rapidly after administration, the specificity for the tagged protein and high sensitivity of the detection method allow detection long after the exogenous protein has been administered. Thus, an abuser cannot claim abnormally elevated production of the endogenous protein, and unlawful use of the tagged protein can be detected. Additionally, the present invention allows the pharmacokinetics and/or pharmacodynamics of the tagged exogenous protein to be detected and monitored.
Therefore, it is an object of the present invention to provide a method for tagging proteins which method enables detection of the exogenous tagged protein over any endogenous polypeptide which may be present in a sample (e.g. such as blood or urine) taken from, for example, a human subject (e.g. an athlete) or other mammalian subject (e.g. domesticated farm livestock).
It is another object of the present invention to provide a modified polypeptide molecule, such as hGH tagged in a manner which is therapeutically acceptable. Further, the tagging method of the present invention enables the biological activity per se of a protein to remain substantially unaltered such that the therapeutic efficacy is maintained and the protein can be administered in a manner identical to or similar with the unmodified protein.
A further specific object of the present invention is to provide a modified hGH molecule substituted with tryptophans at strategic positions in the native hGH sequence.
In a first aspect, the invention provides a method of detecting the presence in a sample of a polypeptide exogenously administered to a mammalian subject from whom the sample is obtained, and distinguishing between such an exogenously administered polypeptide and a naturally-occurring endogenous polypeptide present in the sample; the method comprising obtaining a sample from the subject; and subjecting the sample to analysis of fluorescence at a suitable wavelength; wherein the exogenously administered is tagged with a greater or lesser amount of fluorescence activity, relative to the untagged endogenous polypeptide, at the wavelength(s) analysed.
In a second aspect, the invention provides a composition for administration to a mammalian subject, the composition comprising a polypeptide and a physiologically acceptable carrier substance, characterised in that the polypeptide is tagged with a greater or lesser amount of fluorescent activity relative to an untagged polypeptide endogenously present in the subject, the tagged molecule thereby being distinguishable from the untagged molecule by analysis of the fluorescence characteristics of the respective molecules, excluding those compositions in which the tagged molecule is Growth Hormone and wherein the fluorescent tagging consists solely of one or more of the following substitutions in the tagged Growth Hormone:
G40xe2x86x92Y: F52xe2x86x92Y; W86xe2x86x92F, Y, L, I or V; F103xe2x86x92Y; and 1137xe2x86x92Y.
The tagged molecule is a polypeptide, which may typically be administered to a mammalian subject to exert a beneficial effect (e.g. for clinical or veterinary reasons, or for reasons of animal husbandry). The mammalian subject will generally be human, but may also be a domesticated animal, especially a farm animal such as a bovine, porcine or ovine animal. The tagged molecule will generally therefore be a therapeutic polypeptide (i.e. comprises five or more amino acid residues and has a desirable effect on the subject, with little or no undesired side effect, when administered in an appropriate dose) and will possess the same biological activity as, and normally be substantially identical (except for the tagging) to, a naturally-occurring polypeptide present in the subject, although where the tagged molecule is a recombinant polypeptide it may have additional slight differences relative to the naturally occurring polypeptide (e.g. to increase activity, or to increase stability, e.g. as taught in WO 94/10200). (The xe2x80x9cbiological activityxe2x80x9d of the molecule is that activity by which the molecule exerts its beneficial effect on the subject e.g. stimulation of growth in the case of GH; or stimulation of erythrocyte production in the case of EPO.)
The molecule may be, for example, a pharmaceutical. A particularly preferred molecule is a mammalian growth hormone, especially human growth hormone (hGH), bovine growth hormone (bGH), or porcine growth hormone (pGH); or calcitonin; or erythropoietin (EPO). Accordingly it is preferred that any fluorophores present in the tagged molecule: (a) do not have any significant effect on the biological activity of the molecule; and (b) are essentially non-toxic (that is, any fluorophores present will not cause the tagged molecule to exhibit any toxicity for the subject when the molecule is administered at normal therapeutic doses). Accordingly, tryptophan or tyrosine and closely-related compounds are preferred fluorophores for use in tagged molecules in accordance with the invention. These have the additional advantage of being readily incorporated into polypeptide molecules.
Advantageously, the tagged molecule is either deficient in, or comprises additional, fluorescent entities (fluorophores) relative to the untagged molecule. The tagging may therefore be xe2x80x9cpositivexe2x80x9d (in which the tagged molecule comprises additional fluorophores) or xe2x80x9cnegativexe2x80x9d (where the tagged molecule is deficient in fluorophores relative to the untagged molecule).
As explained above, the naturally occurring amino acid residues tryptophan (W) and, to a lesser extent tyrosine (Y), possess natural fluorophore activity. Thus, if an xe2x80x9cuntaggedxe2x80x9d polypeptide comprises one or more tryptophan and/or tyrosine residues it may be fluorescent. Thus a tagged molecule, in accordance with the invention, may be distinguishable from an untagged molecule by having additional fluorophores (especially if the untagged polypeptide comprises no, or very few, tryptophan or tyrosine residues and thus possesses no, or very little, intrinsic fluorescence). Alternatively, where the untagged molecule comprises a fluorophore (especially a plurality of fluorophores), the tagged molecule may be distinguishable by having fewer fluorophores than the untagged molecule.
Preferably, the tagged molecule comprises additional fluorophores present in amino acid residues or other compounds which are capable of forming a peptide bond, and thus are capable of being covalently incorporated into a polypeptide, either internally during synthesis of the polypeptide, and/or at the C-terminal after synthesis of the bulk of the polypeptide.
Conveniently the fluorophores additionally present in (or absent from) the tagged molecule (relative to the untagged molecule) are tyrosine and/or tryptophan residues, or a synthetic amino acid derivative wherein a fluorophore is covalently joined to an xe2x80x9camino acidxe2x80x9d backbone, the synthetic derivative having the general formula 
wherein R1 comprises the fluorophore and R2 is H, OH, halide or lower alkyl (C1 to C3, substituted or unsubstituted). The fluorophore R1 may be a fluorophore which is present in a naturally occurring amino acid residue (e.g. the aromatic groups of tryptophan or tyrosine) or may be some other fluorophore (typically comprising a delocalised electron system, such as in an aromatic or heterocyclic ring). Such synthetic amino acid derivatives are already known in the art or can readily be prepared using standard organic chemistry techniques.
As a less preferable alternative to the tagged molecule comprising a different number of fluorophores (relative to the untagged molecule), the tagged molecule may comprise the fluorophores at different positionsxe2x80x94the immediate chemical environment can affect the level of fluorescence of a fluorophore. Accordingly, the tagged molecule may not have a different number of fluorophores relative to the untagged molecule, but they may be of different fluorescent activities and/or be differently disposed within the molecule so as to affect their fluorescence.
Where the tagged molecule is a polypeptide, tagging is conveniently accomplished by substituting a non-fluorescent amino acid present in the untagged molecule for an amino acid residue comprising a fluorophore (such as tryptophan, tryosine or a synthetic amino acid derivative), so as to increase the fluorescence of the tagged molecule relative to the untagged molecule.
With the benefit of the teaching of the present specification, and with the benefit of information otherwise readily available as common general knowledge, the person skilled in the art can, by routine trial and error, find appropriate amino acid residues which can be substituted, without substantially affecting the biological activity of the molecule. Conveniently, phenylalanine residues (F) or tyrosine residues (Y) can be replaced with tryptophan residues (W), which exhibit far greater fluorescence activity. Such substitutions are xe2x80x9cconservativexe2x80x9d and thus tend not to have any significant effect on the biological properties of a polypeptide. Further guidance for the person skilled in the art is given in the example below, which utilises principles which are generally applicable to any biologically active polypeptide.
The composition will normally comprise an effective amount of the tagged molecule, such that the biological activity thereof produces a demonstrable effect when administered to the subject. An xe2x80x9ceffective amountxe2x80x9d is the amount of tagged molecule which results in the desired biological effect in the mammalian subject to which the composition is administered. The desired effect will, of course, depend on the identity of the tagged molecule: where the tagged molecule is EPO, for example, the desired effect is an increase in the number of erythrocytes per unit volume of blood in the subject. In some embodiments the composition will be essentially sterile, and suitable for delivery by means of injection (e.g. by transdermal, intravenous, intramuscular or subcutaneous routes). In other embodiments the composition will be in the form of a tablet, pill or capsule (e.g. enteric-coated capsules for slow release) for oral consumption.
Administration of the compositions of the invention into a mammalian subject may be performed according to known methods using any route effective to deliver the required dosage to the subject. Modes of administration include those typically encountered for the species of choice. Because proteins in general are susceptible to degradation in the digestive system, injection is preferred via an intramuscular, transdermal or subcutaneous route. The use of sustained or prolonged release formulations or implants are also suitable modes. Generally, injection of a sustained release formulation is preferred.
The effective dosage range depends on the species, age, weight, and general health of the mammalian subject. These and other parameters which are needed to determine the effective dosage range for a given mammal are well within the purview of one skilled in the art. For instance, in bovines the effective amount of bovine GH (whether tagged or untagged) is in the range of 1.0 to 200 milligrams per animal per day. In pigs, for instance the effective amount of porcine GH is about 60 xcexckg/day.
The physiologically acceptable carrier may be a sterile liquid diluent where the composition is injected (e.g. saline, phosphate-buffered saline, or other aqueous buffer preparation). Where the composition is to be administered orally or transdermally, the carrier may be calcium carbonate, calcium sulphate or other substantially inert solid. Transdermal delivery by means of a needleless injection device may generally be preferred.
Methods of performing the fluorescence analysis may be entirely conventional and well known to those skilled in the art (e.g. spectrofluorimetry). The choice of method will depend in part on the manner in which the exogenous substance is tagged, and the characteristics of the fluorophore (if any) employed in the tagged molecule. For example, where the tagged molecule comprises fewer or more tryptophan residues than the untagged molecule, fluorescence analysis will typically be performed at about 297 nm excitation.
Advantageously the sample is subjected to processing, prior to fluorescence analysis, to enrich or purify the endogenous and (if present) exogenous molecules in the sample. This improves the signal-to-noise ratio. Various methods of enrichment or purification may be employed, using one or more of the following techniques: centrifugation: HPLC: FPLC; affinity chromatography; immunoaffinity chromatography; heat treatment at 50-55xc2x0 C. for ten minutes (this is particularly appropriate for purification of growth hormone, which is relatively heat-stablexe2x80x94contaminating proteins will tend to be denatured, aggregate and precipitate, and so can be simply removed by centrifugation whilst the undenatured growth hormone stays in solution); all of which are well known to those skilled in the art. The preferred method may depend, at least in part, on the identity of the endogenous and exogenous molecules.
The method defined immediately above is extremely useful in detecting the presence of exogenously administered molecules used illicitly by cyclists, athletes and others to improve performance. Very often, such molecules occur naturally (e.g. EPO, hGH, and the like) and are endogenous to the athlete""s body, such that proving illicit use of performance-enhancing substances is very difficult. However, with the benefit of the present invention, such substances can be tagged, and thus made distinguishable over endogenous molecules synthesised naturally in the athlete""s body.
Additionally the invention can be used to monitor the persistence of substances administered to the body. For example, the pharmacokinetics and/or pharmacodynamics of various drugs can readily be monitored by comparing fluorescence activities at different time pointsxe2x80x94this is particularly useful where the tagged drug is otherwise identical to an endogenous compound.
In a preferred embodiment, the tagged molecule is a polypeptide prepared using recombinant DNA technology. In such embodiments the method may additionally comprise the preparation of a nucleic acid sequence encoding the tagged molecule, the sequence being mutated relative to the wild type sequence encoding the untagged molecule. Typically the nucleic acid sequence encoding the tagged polypeptide comprises nucleotide substitutions (relative to the wild type sequence) so as to direct the expression of a polypeptide having one or more tryptophan residues not present in the untagged molecule or, less preferably, directing the expression of a polypeptide having fewer tryptophan residues than in the untagged molecule.
The nucleic acid sequence encoding the tagged molecule may be prepared, for example, by mutation of the wild-type sequence (e.g. by site-directed mutagenesis), by polymerase chain reaction (PCR), or by de novo synthesis (e.g. using an automated DNA synthesiser). All of these techniques are familiar and well-known to those skilled in the art and/or are readily obtained by reference to standard texts in the field (e.g. Sambrook et al, xe2x80x9cMolecular Cloning, A Laboratory Manualxe2x80x9d Cold Spring Harbor Laboratory Press, 1989).
Where the subject is a human, the sample may conveniently be a sample of body fluids, such as a blood, sweat, semen, urine, or saliva sample. Less preferably the sample may be a tissue sample comprising cells (e.g. skin scrapings from the buccal cavity, hair or the like). Where the subject is a domesticated farm animal, the sample may be taken from the animal before or after slaughter. Samples taken after slaughter conveniently include muscle tissue or other solid tissues taken from the carcass.
In another aspect of the present invention there is provided a tagged GH molecule comprising a tryptophan residue substituted for a phenylalanine residue present in a naturally-occurring molecule. In one embodiment, tryptophan is substituted at positions F31 and/or F97 in the amino acid sequence.
In a preferred embodiment, the tagged growth hormone comprises a tryptophan residue at one or more of positions 10, 31, 97, 160 or 176 (of which tryptophan residues at positions 31 and/or 97 are especially preferred). The tagged growth hormone molecule is preferably tagged hGH.
According to a still further aspect of the present invention there is provided a nucleic acid expression vector comprising substantially nucleotides 114-695 of the nucleic acid sequence shown in FIG. 2. The CPG2 signal sequence (nucleotides 39-113) is intended to direct the encoded polypeptide product to the bacterial periplasmxe2x80x94those skilled in the art will appreciate that the CPG3 signal does not form an essential part of the vector, but is useful for expression in prokaryotes. Other signal sequences are well known to those skilled in the art and could be substituted for the CPG2 signal sequence if desired. Thus the expression vector may be designed to cause expression in eukaryotes (e.g. mammalian tissue culture, fungal or yeast cultures) or in prokaryotes (bacterial cultures). In a particular embodiment the expression vector is a prokaryotic expression system, preferably comprising the vector pMTLhGHm described below.